1. Field of the Invention
The invention relates to the detection and quantitation of cyclodextrins and cyclodextrin derivatives in solutions comprising a protein. The invention further relates to methods of evaluating pharmaceutical preparations for the presence of residual cyclodextrins.
2. Background
Cyclodextrins are cyclic polysaccharides most commonly consisting of six (α-cyclodextrin), seven (β-cyclodextrin), or eight (γ-cyclodextrin) glucose units linked by alpha-1,4-glucosidic bonds in a donut shaped ring. As a consequence of the chair formation of the sugar units, all secondary hydroxyl units are located on one side of the ring, while all of the primary hydroxyl groups are situated on the other side. As a result, the external faces are hydrophilic, making the cyclodextrins water-soluble. In contrast, the cavities of the cyclodextrins are hydrophobic, since they are lined by the hydrogens of atoms C3 and C5 and by ether-like oxygens. This structure allows cyclodextrins to form inclusion complexes with hydrophobic compounds. Based on this characteristic, cyclodextrins are widely used to increase the solubility of poorly water soluble pharmaceuticals, enhance pharmaceutical stability, and reduce unwanted side effects of pharmaceuticals.
The hydroxyl groups of cyclodextrins may be derivatized to alter the characteristics of the molecule (e.g., water solubility, binding specificity). Methyl-β-cyclodextrin (MBCD) is one of the most frequently used cyclodextrin derivatives.
Cyclodextrins are also used in cell culture systems to carry hydrophobic nutrients, such as cholesterol, into cells. Cyclodextrins are particularly useful for the culturing of cholesterol auxotrophic cells, e.g., cholesterol auxotrophic CHO cells, COS cells, and NSO cells. When cell cultures are used to produce naturally occurring or recombinant proteins, particularly therapeutic proteins, the presence of cyclodextrins as a residual contaminant in the purified protein product is an issue in the production of pharmaceutical products. Thus, a sensitive and rapid technique for the detection and quantification of low levels of cyclodextrins or cyclodextrin derivatives in a solution comprising a protein is needed.
Grosse et al. (J. Chromatography B 694:219 (1997)) disclose a technique for detecting MBCD using size exclusion chromatography in the presence of a fluorophore (1-naphthol) that forms an inclusion complex with MBCD and allows fluorescent detection of the inclusion complex. This technique, which was designed for the detection of MBCD in plasma samples for pharmacokinetic studies, requires extraction of samples with organic solvents, evaporation, and dissolution of the residue in the mobile phase prior to chromatography. Gage et al. (J. Pharm. Biomed. Analysis 22773 (2000)) disclose a similar technique using 1-naphthol for measuring the presence of sulphobutylether-β-cyclodextrin in plasma samples. This method requires processing of the sample, including solid phase extraction, evaporation, and dissolution of the residue, prior to chromatography. These techniques do not achieve separation of cyclodextrins from protein components.
A need exists in the art for a method of testing for the presence of cyclodextrin or cyclodextrin derivatives in the presence of a protein, preferably without the need for sample extraction steps.